Food and Drug Administration (FDA) regulations recommend that pharmaceutical manufacturers using recombinant DNA processes demonstrate that products contain very low levels of DNA. Current FDA guidelines specify that levels of DNA amounting to less than 10 picograms of DNA per dose should generally be acceptable. Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals, Office of Biologics Research and Review, FDA (Nov. 18, 1987).
Current methods for determining the levels of residual DNA in protein product include the Threshold.TM. Total DNA Assay system (Molecular Devices Corporation, Menlo Park, Calif.), which uses an automated reader to quantitate DNA by detecting the rate of pH change in enzyme-bound DNA samples. This method is disadvantageous in that it is costly, labor intensive, and is restrictive in that specific compatible buffers must be chosen. Further, because of bulk drug substance formulations and assay system volume restrictions which limit sensitivity, a full dose cannot always be tested using this assay.
The polymerase chain reaction (PCR), Mullis et al., Methods in Enzymology, 155:335 (1987) allows nucleic acid sequences up to a few kilobases in length to be amplified in large amounts in a relatively short period of time. Generally, oligonucleotides having DNA sequences directed to a known DNA sequence in a target DNA molecule are used as primers for amplification.
Silver et at., U.S. Pat. 5,104,792, discloses a method by which, using a collection of "universal primers", PCR can be used to amplify viral nucleic acids present in small amounts in clinical material.
Goldman et al., Clinical Chemistry, 37:1523 (1991) describe a method by which primers developed to amplify the E. coli gene for 16S ribosomal RNA, which is repeated about seven times in the E. coli genome, are used as a marker for E. coli DNA.
These known processes, however, suffer the disadvantages of requiring DNA primers which are specific to genes present in the host organism genome only in small numbers, and therefore are not capable of being used to detect the presence of DNA contamination on the order of about 1 to 10 picogram. Because of the volume and formulation restrictions, there exists a need for more sensitive methods of detecting DNA contamination present in small amounts, on the order of at least about 0.01 to about 0.1 picogram.